I. NEMO Syndrome NEMO, a non-catalytically active component of the IkappaB alpha kinase (IKK) complex, is required for IKK enzymatic activity and localization. In its absence nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcription factor family members do not translocate to the nucleus to regulate gene transcription, and normal development of the organism, including the immune system, does not occur. We apply a forward genetic approach as the basis of our analysis, in that specific mutations which result in NEMO immunodeficiency and inflammatory disease are investigated using biochemical, cell biological, and molecular techniques. In addition to clinical and laboratory evaluation of human patients these studies are conducted in a reconstitution system utilizing NEMO-deficient Jurkat T cells. NEMO not only is required for IKK activation, but is also thought to serve as a scaffold to other signaling proteins. Its regulation is complex: splice isoforms exist, and it is post-translationally modified by serine phosphorylation, non-degradative ubiquitination, sumoylation. We recently found that particular C-terminal truncation mutations that increase NF-kB activation do not do so by increasing IKK kinase activity, suggesting impairment in a secondary regulatory function. In addition to being directly modified, NEMO recognizes different ubiquitin chains via its central UBAN motif and the C-terminal Zinc Finger (ZF). C terminal truncation has been shown in vitro to shift the affinity of the centrally located UBAN ubiquitin binding motif from K63 to linear ubiquitin. We have developed an expression system to express mutant forms of NEMO and are currently working to determine qualitative binding affinities in different forms of mutant NEMO that result in inflammatory disease. We hope to gain insight into this potential affinity switch mechanism which may determine how signaling via distinct receptors such as Toll-like receptors and TNFR can induce different response, and result in inflammation. Variability of clinical disease phenotypes and immune-function lab abnormalities make management of individuals with NEMO syndrome and inflammatory disease difficult. Because of our incomplete understanding, treatment paradigms, such as the use of allogeneic hematopoietic stem cell transplantation, cannot at this point be standardized and often result in poor outcomes. We are establishing protocols to evaluate patients at the Clinical Center to be able to comprehensively characterize the spectrum of phenotypes, and are working to develop protocols to offer advanced treatment based on our clinical and laboratory findings. Because NEMO syndrome is associated with defects in development of ectodermal structures in addition to secondary lymphoid structures in association with certain mutations, we seek to understand the function of NEMO in both of these roles. Therefore, we are developing techniques to harness iPS cell technologies to study the effects of mutation on the development and function of various cell types. II. JAK2 haploinsufficiency NF-kB signaling defects affect other immune signaling pathways and conversely mutations in other pathways have been shown to impact NF-kB-dependent activation. For example, a negative regulator of STAT3 signaling, SOCS-3, is a target gene upregulated by IKK-dependendent NF-kB activation. An individual with a chromosomal alteration leading to loss of one copy of JAK2 was recently identified and has defects in T and B memory cell development, cytopenias, and immunoglobulin production, some of which overlaps with phenotypic characteristics of NEMO syndrome. We are currently investigating the signaling in EBV transformed B cells and primary cells from the patient and have found that Interleukin 6 mediated STAT1 and STAT3 phosphorylation is reduced; further work is being done to determine the scope of the related signaling defects. Additionally, we are developing methods to generate iPS cells to study the effects of this mutation in different cell types of the immune system. III. WASp role in iTreg signaling Recent work indicates a role for the inhibitor of NF-kappaB kinase complex member, IKKbeta, in the subcellular localization of signaling proteins (HOMER-3) which mediate TCR signaling through mediating cystoskeletal changes and whose normal function is required to prevent autoimmunity. Recently, work has shown that iTreg development is abnormal in individuals with WAS mutations, and that c-Rel is required for normal development of this subset. Work is currently being done to evaluate the role of WASp deletion in mice on iTreg development and NF-kB function. In WAS deficient iTreg, we have found that CD3 stimulation results in 20-fold higher c-Rel cytoplasmic levels compared to normal control, but that this does not lead to increased c-Rel nuclear levels or binding following costimulation with CD28. We will continue to investigate how WASp deficiency leads to NF-kB signaling defects and how this influences T cell subset development and function.